Nuclear matrix proteins

ABSTRACT

Nuclear matrix proteins (NMP) which are characterized by a defined expression in tissue are provided. These NMPs are useful markers in diagnosing and monitoring the stage of malignancy of a cell and treating cell proliferative disorders associated with the NMP. Also provided are substantially purified polypeptides and nucleotide sequences encoding the NMPs of the invention.

[0001] This is a continuation-in-part U.S. patent application Ser. No.08/015,624, filed Feb. 9, 1993.

[0002] This invention was made with Government support under NIH SPOREGrant P50 CA 58236-01 awarded by the National Institutes of Health,DK-19300 National Institute of Arthritis, Diabetes and Digestive andKidney Diseases and CA 15416, National Cancer Institute.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] This invention relates generally to nuclear matrix proteins andspecifically to novel nuclear matrix proteins with defined tissueexpression patterns in normal cells and cells associated with cellproliferative disorders.

[0005] 2. Description of Related Art

[0006] Advances in recombinant DNA technology have led to the discoveryof normal cellular genes (proto-oncogenes and tumor suppressor genes)which control growth, development, and differentiation. Under certaincircumstances, regulation of these genes is altered and normal cellsassume neoplastic growth behavior. In some cases, the normal cellphenotype can be restored by various manipulations associated with thesegenes. There are over 40 known proto-oncogenes and suppressor genes todate, which fall into various categories depending on their functionalcharacteristics. These include, 1) growth factors and growth factorreceptors, 2) messengers of intracellular signal transduction pathways,for example, between the cytoplasm and the nucleus, and 3) regulatoryproteins influencing gene expression and DNA replication, located bothwithin and outside the nucleus.

[0007] During their life span, normal cells begin in an immature statewith proliferative potential, pass through sequential stages ofdifferentiation, and eventually end in cell death. Cancer, on the otherhand, is a multistep process which can be defined in terms of stages ofmalignancy wherein the normal orderly progression is aberrant, probablydue to alterations in oncogenes, tumor suppressor genes, and othergenes. Research on oncogenes and their products has led to a morefundamental understanding of the mechanisms of cancer causation andmaintenance allowing more rational means of diagnosing and treatingmalignancies.

[0008] Genes associated with the control of normal growth anddifferentiation of cells include genes which encode regulatory proteinswhich influence gene expression and DNA replication. The gene productsof many of these genes localize in the nucleus and many are DNA bindingproteins. The nucleus of an animal cell contains cellular DNA complexedwith protein, referred to as chromatin. The chromatin is organized bythe internal skeleton of the nucleus, called the nuclear matrix. Nuclearmatrix proteins (NMP) associated with DNA may be growth/differentiationregulatory proteins which play a role in the regulation of geneexpression in a cell. In cells that have lost their growth regulatorymechanisms, it can be envisioned that a nucleus-specific protein maycontinuously activate a transcriptional promoter region of a gene,causing over-expression of the gene. Similarly, a nuclear protein whichfunctions as a suppressor to control or suppress the expression ofvarious proto-oncogenes, may be under-expressed or expressed in a mutantform, thereby allowing aberrant expression of a gene which otherwisewould be suppressed.

[0009] Current cancer tests are generally nonspecific, insensitive and,consequently, of limited clinical application. For example, abiochemical test, widely used for both diagnostic and monitoring ofcancer, measures levels of carcinoembryonic antigen (CEA). CEA is anoncofetal antigen detectable in large amounts in embryonal tissue, butin small amounts in normal adult tissues. Serum of patients with certaingastrointestinal cancers contains elevated CEA levels that can bemeasured by immunological methods. The amount of CEA in serum correlateswith the remission or relapse of these tumors, with the levelsdecreasing abruptly after surgical removal of the tumor. The return ofelevated CEA levels signifies a return of malignant cells. CEA, however,is also a normal glycoprotein found at low levels in nearly all adults.Moreover, this protein can be elevated with several nonmalignantconditions and is not elevated in the presence of many cancers.Therefore it is far from ideal as a cancer marker. A similar oncofetaltumor marker is alpha-fetoprotein, an embryonic form of albumin. Again,the antigen is detectable in high amounts in embryonal tissue and in lowamounts in normal adults. It is elevated in a number of gastrointestinalmalignancies including hepatoma. Like CEA, a decrease correlates withthe remission of cancer and a re-elevation with relapse. There isinsufficient sensitivity and specificity to make this marker useful forscreening for malignancy or for monitoring previously diagnosed cancerin any but a few selected cases.

[0010] In view of the foregoing, there remains a need for new cancermarkers which would allow more effective diagnosis, prognosis andtreatment regimes. The identification of NMPs which are associated withthe regulation of gene expression or cellular structure in normal andcancer cells would provide ideal markers for identification of the stageof malignancy of a cell.

SUMMARY OF THE INVENTION

[0011] The present invention is based in the discovery of novel nuclearmatrix proteins (NMP) which have defined patterns of tissue expressionin different stages of abnormal growth and malignancy of a cell. TheNMPs of the invention are defined by a tissue expression patterncharacteristic of any one of the proteins, NPB-1, NPB-2, NPB-3, NPB-4,NPB-5, NPB-6, NPB-7, NP-1, NP-2, NP-3, BPC-1, BPC-2, BPC-3, and PC-1.These proteins were initially identified as being associated with normalprostate tissue (NP), both normal and benign hyperplasia prostate tissue(NBP), benign hyperplasia and cancerous prostate tissue (BPC), orprostate cancer tissue (PC).

[0012] The invention provides nucleotides which encode the novel NMPs.The NMPs of the invention provide the basis for a method of detecting acell proliferative disorder in a subject comprising contacting acellular component with a reagent which binds to the NMP. The method isespecially useful for detecting a cell proliferative disorder in atissue of the urogenital system, and specifically the prostate.

[0013] The invention also provides a method of treating a cellproliferative disorder associated with NPB-1, NPB-2, NPB-3, NPB-4,NPB-5, NPB-6, NPB-7, NP-1, NP-2, NP-3, BPC-1, BPC-2, BPC-3, and PC-1.Such a method may include gene therapy, for example.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows nuclear matrix protein composition of normal humanprostate (A), benign prostatic hyperplasia (B) and prostate cancer (C).Large arrow panel A. variable group of proteins that were inconsistentlypresent on various types of tissue. Small arrows, proteins that areconsistently changed in all tissues and are identified by molecularweight and isoelectric point in table 1. Abbreviations: LA—lamin A,LB—lamin B, LC—lamin C, A—actin, NP—normal prostate, NPB—normal prostateand BPH, BPC—BPH and prostate cancer, PC-prostate cancer, kD—molecularweight in thousands, SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and pl—isoelectric point.

[0015]FIG. 2 shows specific nuclear matrix proteins in BPH and prostatecancer. Schematic of major tissue specific nuclear matrix proteins ofnormal prostate, BPH and prostate cancer. Abbreviations: kD—molecularweight in thousands, SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gelelectrophoresis and pl—isoelectric point and BPH—benign prostatichyperplasia.

[0016]FIG. 3 shows two models of multistep progression from normalprostate (Normal) to benign prostatic hyperplasia (BPH) or to prostatecancer (Cancer). Model I predicts that similar events occur in bothpathways. Model II predicts different events occurring when progressingfrom normal to BPH as when progressing to cancer.

DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides substantially pure nuclear matrixproteins (NMP) or functional fragments thereof, wherein the protein hasa tissue expression pattern characteristic of a protein selected fromthe group comprising NPB-1, NPB-2, NPB-3, NPB-4, NPB-5, NPB-6, NPB-7,NP-1, NP-2, NP-3, BPC-1, BPC-2, BPC-3, and PC-1. The invention alsoprovides polynucleotide sequences which encode these proteins. The NMPsof the invention can generally be characterized by their presence in acell during a specific stage of a cell proliferative disorder.

[0018] The term “cell-proliferative disorder” denotes malignant as wellas non-malignant cell populations which often appear to differ from thesurrounding tissue both morphologically and genotypically. Malignancy(i.e, cancer) is a multistep process and the proteins of the inventionare associated with three broad steps in the transition from a normalcell to a cancer cell. In broad stages, normal tissue (stage 1) maybegin to show signs of hyperplasia (stage 2) or show signs of neoplasia(stage 3). As used herein, “hyperplasia” refers to cells which exhibitabnormal multiplication or abnormal arrangement in a tissue. Included inthe term hyperplasia, are benign cellular proliferative disorders,including benign tumors. Proteins of the invention which exhibit atissue expression pattern in both normal tissue and benign hyperplastictissue (NPB) (non-malignant) include NPB-1, NPB-2, NPB-3, NPB4, NPB-5,NPB-6 and NPB-7. The term “tissue expression pattern” refers to thesynthesis of a gene product of an NMP gene at a level which isdetectable by methods commonly used by those of skill in the art (e.g.,SDS-polyacrylamide gel electrophoresis). As used herein, “neoplasia”refers to abnormal new growth, which results in a tumor. Unlikehyperplasia, neoplastic proliferation persists even in the absence ofthe original stimulus and characterized as uncontrolled and progressive.Malignant neoplasms, or malignant tumors, are distinguished from benigntumors in that the former show a greater degree of anaplasia and havethe properties of invasion and metastasis. A protein of the invention,PC-1, is an example of a protein which has an expression pattern seen inmalignant neoplasms. The proteins of the invention, BPC-1, BPC-2 andBPC-3 are examples of proteins which are expressed in both benignhyperplastic tissue or tumors and also neoplastic tissues or tumors. Onthe other hand, proteins NP-1, NP-2 and NP-3 have tissue expressionpatterns in normal (non-tumor), non-hyperplastic, and non-neoplastictissue. Thus, the presence of these latter proteins essentially rule outthe presence of a cell proliferative disorder.

[0019] In summary, BPC-1, BPC-2, and BPC-3 and PC-1 are associated withtumor cells; NPB-1, NPB-2, NPB-3, NPB4, NPB-5, NPB-6, and NPB-7 areassociated with normal and non-malignant hyperplastic or tumor cells;PC-1 is associated with malignant cells; and NP-1, NP-2, and NP-3 areassociated with non-tumor cells. The term “associated with” refers tothe correlation between the expression pattern of the protein and thestage of progression to cancer.

[0020] The invention provides substantially pure NMPs or functionalfragments thereof. As used herein, the term “functional polypeptide” or“functional fragment” refers to a polypeptide which possesses abiological function or activity which is identified through a definedfunctional assay and which is associated with a particular biologic,morphologic or phenotypic alteration in the cell. The polypeptidefragment possessing biological function can vary from as small as anepitope to which an antibody molecule can bind to as large as apolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell. It is understood thatall functional polypeptides encoding all or a functional portion of theNMPs of the invention are also included herein, so long as they arefound in the nuclear matrix and exhibit the tissue expression patterncharacteristic of a given NMP of the invention. A “functionalpolynucleotide” denotes a polynucleotide which encodes a functionalpolypeptide as described herein.

[0021] The term “substantially pure” or “isolated” means any NMPfunctional polypeptide of the present invention, or any gene encoding anNMP functional polypeptide, which is essentially free of otherpolypeptides or genes, respectively, or of other contaminants with whichit might normally be found in nature, and as such exists in a form notfound in nature. The invention also provides functional derivatives ofthe NMPs of the invention. By “functional derivative” is meant the“fragments,” “variants,” “analogues,” or “chemical derivatives” of amolecule. A “fragment” of a molecule, such as any of the DNA or aminoacid sequences of the present invention, includes any nucleotide oramino acid subset of the molecule. A “variant” of such molecule refersto a naturally occurring molecule substantially similar to either theentire molecule, or a fragment thereof. An “analog” of a molecule refersto a non-natural molecule substantially similar to either the entiremolecule or a fragment thereof.

[0022] Procedures which may be used to isolate the NMPs of the inventioninclude those commonly used for the separation of protein substancesincluding, for example, treatment of a sample containing NMP with commonprecipitants for proteins, followed by fractionation techniques such asion exchange chromatography, affinity chromatography, molecular sievechromatography, adsorption chromatography, ultrafiltration and variouscombinations thereof. The NMPs can be purified from a cell suspension bymethods described in U.S. Pat. Nos. 4,885,236 and 4,882,268, forexample. Other methods for purification of the polypeptides of theinvention will be known to those of skill in the art (see, for example,Current Protocols in Immunology, Coligan, et al, eds. 1992, incorporatedherein by reference).

[0023] The NMP containing fractions can be subjected to SDS PAGE undersuitable conditions and the gel slice containing NMP activity orcorresponding to the molecular weight of the NMP of interest isrecovered. SDS PAGE is performed according to the method of Laemmli, etal., (Nature, 227:680, 1970) and is a technique well known to those inthe art. Variations in conditions which are within a suitable range areunderstood to be encompassed within the purification procedure.

[0024] The NMP containing fraction from the SDS PAGE is subjected toreverse phase HPLC and eluted with acetonitrile for example. The NMPwhich is obtained is substantially pure to permit N-terminal amino acidsequencing. The solution is dried under vacuum and redissolved in asmall volume of acetonitrile 95%+TFA (0.08%). The concentrated sample isthen introduced in a sequencer connected to a phenylthiohydantoine (PTH)analyzer.

[0025] The invention provides polynucleotides, such as DNA, cDNA, andRNA, encoding novel NMP functional polypeptides. It is understood thatall polynucleotides encoding all or a functional portion of the NMPs ofthe invention are also included herein, so long as they are found in thenuclear matrix and exhibit the tissue expression pattern characteristicof a given NMP of the invention. Such polynucleotides include bothnaturally occurring and intentionally manipulated, for example,mutagenized polynucleotides.

[0026] The polynucleotide sequence for NMP also includes antisensesequences and sequences that are degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, as long as the amino acid sequence ofNMP results in a functional polypeptide (at least, in the case of thesense polynucleotide strand), all degenerate nucleotide sequences areincluded in the invention. Where the antisense polynucleotide isconcerned, the invention embraces all antisense polynucleotides capableof inhibiting production of the NMP polypeptide.

[0027] DNA sequences of the invention can be obtained by severalmethods. For example, the DNA can be isolated using hybridizationprocedures which are well known in the art. These include, but are notlimited to: 1) hybridization of probes to genomic or cDNA libraries todetect shared nucleotide sequences; 2) antibody screening of expressionlibraries to detect shared structural features; and 3) synthesis by thepolymerase chain reaction (PCR). RNA sequences of the invention can beobtained by methods known in the art (See for example, Current Protocolsin Molecular Biology, Ausubel, et al. eds., 1989, incorporated herein byreference).

[0028] The development of specific DNA sequences encoding NMPs of theinvention can be obtained by: (1) isolation of a double-stranded DNAsequence from the genomic DNA; (2) chemical manufacture of a DNAsequence to provide the necessary codons for the polypeptide ofinterest; and (3) in vitro synthesis of a double-stranded DNA sequenceby reverse transcription of mRNA isolated from a eukaryotic donor cell.In the latter case, a double-stranded DNA complement of mRNA iseventually formed which is generally referred to as cDNA. Of these threemethods for developing specific DNA sequences for use in recombinantprocedures, the isolation of genomic DNA isolates is the least common.This is especially true when it is desirable to obtain the microbialexpression of mammalian polypeptides due to the presence of introns.

[0029] The synthesis of DNA sequences is frequently the method of choicewhen the entire sequence of amino acid residues of the desiredpolypeptide product is known. When the entire sequence of amino acidresidues of the desired polypeptide is not known, the direct synthesisof DNA sequences is not possible and the method of choice is theformation of cDNA sequences. Among the standard procedures for isolatingcDNA sequences of interest is the formation of plasmid-carrying cDNAlibraries which are derived from reverse transcription of mRNA which isabundant in donor cells that have a high level of genetic expression.When used in combination with polymerase chain reaction technology, evenrare expression products can be cloned. In those cases where significantportions of the amino acid sequence of the polypeptide are known, theproduction of labeled single or double-stranded DNA or RNA probesequences duplicating a sequence putatively present in the target cDNAmay be employed in DNA/DNA hybridization procedures which are carriedout on cloned copies of the cDNA which have been denatured into asingle-stranded form (Jay, et al., Nucleic Acid Research, 11:2325,1983).

[0030] Hybridization procedures are useful for the screening ofrecombinant clones by using labeled mixed synthetic oligonucleotideprobes where each probe is potentially the complete complement of aspecific DNA sequence in the hybridization sample which includes aheterogeneous mixture of denatured double-stranded DNA. For suchscreening, hybridization is preferably performed on eithersingle-stranded DNA or denatured double-stranded DNA. Hybridization isparticularly useful in the detection of cDNA clones derived from sourceswhere an extremely low amount of mRNA sequences relating to thepolypeptide of interest are present. By using stringent hybridizationconditions directed to avoid non-specific binding, it is possible, forexample, to allow the autoradiographic visualization of a specific cDNAclone by the hybridization of the target DNA to that single probe in themixture which is its complete complement (Wallace, et al, Nucleic AcidResearch, 9:879, 1981).

[0031] An NMP-containing cDNA library can be screened by injecting thevarious cDNAs into oocytes, allowing sufficient time for expression ofthe cDNA gene products to occur, and testing for the presence of thedesired cDNA expression product, for example, by using an antibodyspecific for NMP polypeptide or by using functional assays for NMPactivity and a tissue expression pattern characteristic of NMP.

[0032] Screening procedures which rely on nucleic acid hybridizationmake it possible to isolate any gene sequence from any organism,provided the appropriate probe is available. Oligonucleotide probes,which correspond to a part of the sequence encoding the protein inquestion, can be synthesized chemically. This requires that short,oligopeptide stretches of amino acid sequence must be known. The DNAsequence encoding the protein can be deduced from the genetic code,however, the degeneracy of the code must be taken into account. It ispossible to perform a mixed addition reaction which utilizes aheterogeneous mixture of denatured double-stranded DNA when the sequenceis degenerate. For such screening, hybridization is preferably performedon either single-stranded DNA or denatured double-stranded DNA.

[0033] Since the novel DNA sequences of the invention encode essentiallyall or part of an NMP, it is now a routine matter to prepare, subclone,and express smaller polypeptide fragments of DNA from this orcorresponding DNA sequences. Alternatively, by utilizing the DNAfragments disclosed herein which define the unique NMP classes of theinvention it is possible, in conjunction with known techniques, todetermine the DNA sequences encoding the entire NMP. Such techniques aredescribed in U.S. Pat. No. 4,394,443 and U.S. Pat. No. 4,446,235 whichare incorporated herein by reference.

[0034] A cDNA expression library, such as lambda gt 11, can be screenedindirectly for NMP peptides having at least one epitope, usingantibodies specific for NMP. Such antibodies can be either polyclonallyor monoclonally derived and used to detect expression product indicativeof the presence of NMP cDNA.

[0035] Polyclonal antibodies are prepared by immunization of an animal,e.g., rabbit, with an immunogenic sample of NMP followed by purificationof the antibody by methods well known in the art.

[0036] Antibodies provided in the present invention are immunoreactivewith the NMP of the invention. Antibody which consists essentially ofpooled monoclonal antibodies with different epitopic specificities, aswell as distinct monoclonal antibody preparations are provided.Monoclonal antibodies are made from antigen containing fragments of theprotein by methods well known in the art (Kohler, et al., Nature,256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al.,ed., 1989).

[0037] The antibodies of the invention can be used in immunoaffinitychromatography for the isolation of sequences containing an NMP of thepresent invention. One way by which such immunoaffinity chromatographycan be utilized is by the binding of the antibodies of the invention toCNBr-Sepharose-4B or Tresyl activated Sepharose (Pharmacia). These solidphase-bound antibodies can then be used to specifically bind sequencescontaining NMP from mixtures of other proteins to enable isolation andpurification thereof. Bound NMP sequences can be eluted from theaffinity chromatographic material using techniques known to those ofordinary skill in the art such as, for example, chaotropic agents, lowpH, or urea.

[0038] The production of an NMP DNA sequence can be accomplished byoligonucleotide(s) which are primers for amplification of the genomicpolynucleotide encoding an NMP. These unique oligonucleotide primers canbe produced based upon identification of the flanking regions contiguouswith the polynucleotide encoding the NMP. These oligonucleotide primerscomprise sequences which are capable of hybridizing with the flankingnucleotide sequence encoding an NMP polypeptide and sequencescomplementary thereto.

[0039] The primers of the invention include oligonucleotides ofsufficient length and appropriate sequence so as to provide specificinitiation of polymerization on a significant number of nucleic acids inthe polynucleotide encoding the NMP. Specifically, the term “primer” asused herein refers to a sequence comprising two or moredeoxyribonucleotides or ribonucleotides, preferably more than three,which sequence is capable of initiating synthesis of a primer extensionproduct, which is substantially complementary to an NMP strand.Experimental conditions conducive to synthesis include the presence ofnucleoside triphosphates and an agent for polymerization and extension,such as DNA polymerase, and a suitable temperature and pH. The primer ispreferably single stranded for maximum efficiency in amplification, butmay be double stranded. If double stranded, the primer is first treatedto separate the two strands before being used to prepare extensionproducts. Preferably, the primer is an oligodeoxyribonucleotide. Theprimer must be sufficiently long to prime the synthesis of extensionproducts in the presence of the inducing agent for polymerization andextension of the nucleotides. The exact length of primer will depend onmany factors, including temperature, buffer, and nucleotide composition.The oligonucleotide primer typically contains 15-22 or more nucleotides,although it may contain fewer nucleotides.

[0040] Primers of the invention are designed to be “substantially”complementary to each strand of polynucleotide encoding the NMP to beamplified. This means that the primers must be sufficientlycomplementary to hybridize with their respective strands underconditions which allow the agent for polymerization and nucleotideextension to act. In other words, the primers should have sufficientcomplementarity with the flanking sequences to hybridize therewith andpermit amplification of the polynucleotide encoding the receptor NMP.Preferably, the primers have exact complementarity with the flankingsequence strand.

[0041] Oligonucleotide primers of the invention are employed in theamplification process which is an enzymatic chain reaction that producesexponential quantities of polynucleotide encoding the NMP relative tothe number of reaction steps involved. Typically, one primer iscomplementary to the negative (−) strand of the polynucleotide encodingthe NMP and the other is complementary to the positive (+) strand.Annealing the primers to denatured nucleic acid followed by extensionwith an enzyme, such as the large fragment of DNA Polymerase I (Klenow)and nucleotides, results in newly synthesized (+) and (−) strandscontaining the NMP sequence. Because these newly synthesized sequencesare also templates, repeated cycles of denaturing, primer annealing, andextension results in exponential production of the sequence (i.e., theNMP polynucleotide sequence) defined by the primer. The product of thechain reaction is a discrete nucleic acid duplex with terminicorresponding to the ends of the specific primers employed. Those ofskill in the art will know of other amplification methodologies whichcan also be utilized to increase the copy number of target nucleic acid.These may include, for example, ligation activated transcription (LAT),ligase chain reaction (LCR), and strand displacement activation (SDA),although PCR is the preferred method.

[0042] The oligonucleotide primers of the invention may be preparedusing any suitable method, such as conventional phosphotriester andphosphodiester methods or automated embodiments thereof. In one suchautomated embodiment, diethylphosphoramidites are used as startingmaterials and may be synthesized as described by Beaucage, et al.(Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizingoligonucleotides on a modified solid support is described in U.S. Pat.No. 4,458,066. One method of amplification which can be used accordingto this invention is the polymerase chain reaction (PCR) described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

[0043] Any nucleic acid specimen, in purified or nonpurified form, canbe utilized as the starting nucleic acid for the above procedures,provided it contains, or is suspected of containing, the specificnucleic acid sequence of an NMP of the invention. Thus, the process mayemploy, for example, DNA or RNA (including mRNA), wherein DNA or RNA maybe single stranded or double stranded. In the event that RNA is to beused as a template, enzymes, and/or conditions optimal for reversetranscribing the template to DNA would be utilized. In addition, aDNA-RNA hybrid which contains one strand of each may be utilized. Amixture of nucleic acids may also be employed, or the nucleic acidsproduced in a previous amplification reaction herein, using the same ordifferent primers may be so utilized. The specific nucleic acid sequenceto be amplified, (i.e., NMP sequence), may be a fraction of a largermolecule or can be present initially as a discrete molecule, so that thespecific sequence constitutes the entire nucleic acid. It is notnecessary that the sequence to be amplified be present initially in apure form; it may be a minor fraction of a complex mixture, such ascontained in whole human DNA.

[0044] DNA or RNA utilized herein may be extracted from a body sample,such as prostate tissue, or various other tissue, by a variety oftechniques such as that described by Maniatis, et al. (MolecularCloning, 280:281, 1982). If the extracted sample is impure (such asplasma, serum, ejaculate or blood), it may be treated beforeamplification with an amount of a reagent effective to open the cells,fluids, tissues, or animal cell membranes of the sample, and to exposeand/or separate the strand(s) of the nucleic acid(s). This lysing andnucleic acid denaturing step to expose and separate the strands willallow amplification to occur much more readily.

[0045] Where the target nucleic acid sequence of the sample contains twostrands, it is necessary to separate the strands of the nucleic acidbefore it can be used as the template. Strand separation can be effectedeither as a separate step or simultaneously with the synthesis of theprimer extension products. This strand separation can be accomplishedusing various suitable denaturing conditions, including physical,chemical, or enzymatic means, the word “denaturing” includes all suchmeans. One physical method of separating nucleic acid strands involvesheating the nucleic acid until it is denatured. Typical heatdenaturation may involve temperatures ranging from about 80° to 105° C.for times ranging from about 1 to 10 minutes. Strand separation may alsobe induced by an enzyme from the class of enzymes known as helicases orby the enzyme RecA, which has helicase activity, and in the presence ofriboATP, is known to denature DNA. The reaction conditions suitable forstrand separation of nucleic acids with helicases are described by KuhnHoffmann-Berling (CSH-Quantitative Biology, 43:63, 1978) and techniquesfor using RecA are reviewed in C. Radding (Ann. Rev. Genetics,16:405-437, 1982).

[0046] If the nucleic acid containing the sequence to be amplified issingle stranded, its complement is synthesized by adding one or twooligonucleotide primers. If a single primer is utilized, a primerextension product is synthesized in the presence of primer, an agent forpolymerization, and the four nucleoside triphosphates described below.The product will be partially complementary to the single-strandednucleic acid and will hybridize with a single-stranded nucleic acid toform a duplex of unequal length strands that may then be separated intosingle strands to produce two single separated complementary strands.Alternatively, two primers may be added to the single-stranded nucleicacid and the reaction carried out as described.

[0047] When complementary strands of nucleic acid or acids areseparated, regardless of whether the nucleic acid was originally doubleor single stranded, the separated strands are ready to be used as atemplate for the synthesis of additional nucleic acid strands. Thissynthesis is performed under conditions allowing hybridization ofprimers to templates to occur. Generally synthesis occurs in a bufferedaqueous solution, preferably at a pH of 7-9, most preferably about 8.Preferably, a molar excess (for genomic nucleic acid, usually about10⁸:1 primer:template) of the two oligonucleotide primers is added tothe buffer containing the separated template strands. It is understood,however, that the amount of complementary strand may not be known if theprocess of the invention is used for diagnostic applications, so thatthe amount of primer relative to the amount of complementary strandcannot be determined with certainty. As a practical matter, however, theamount of primer added will generally be in molar excess over the amountof complementary strand (template) when the sequence to be amplified iscontained in a mixture of complicated long-chain nucleic acid strands. Alarge molar excess is preferred to improve the efficiency of theprocess.

[0048] The deoxyribonucleotide triphosphates dATP , dCTP, dGTP, and dTTPare added to the synthesis mixture, either separately or together withthe primers, in adequate amounts and the resulting solution is heated toabout 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period, the solution is allowed to cool toroom temperature, which is preferable for the primer hybridization. Tothe cooled mixture is added an appropriate agent for effecting theprimer extension reaction (called herein “agent for polymerization”),and the reaction is allowed to occur under conditions known in the art.The agent for polymerization may also be added together with the otherreagents if it is heat stable. This synthesis (or amplification)reaction may occur at room temperature up to a temperature above whichthe agent for polymerization no longer functions. Thus, for example, ifDNA polymerase is used as the agent, the temperature is generally nogreater than about 40° C.. Most conveniently the reaction occurs at roomtemperature.

[0049] The agent for polymerization may be any compound or system whichwill function to accomplish the synthesis of primer extension products,including enzymes.

[0050] Suitable enzymes for this purpose include, for example, E. coliDNA polymerase l, Klenow fragment of E. coli DNA polymerase I, T4 DNApolymerase, other available DNA polymerases, polymerase muteins, reversetranscriptase, and other enzymes, including heat-stable enzymes (i.e.,those enzymes which perform primer extension after being subjected totemperatures sufficiently elevated to cause denaturation). Suitableenzymes will facilitate combination of the nucleotides in the propermanner to form the primer extension products which are complementary toeach NMP nucleic acid strand. Generally, the synthesis will be initiatedat the 3′ end of each primer and proceed in the 5′ direction along thetemplate strand, until synthesis terminates, producing molecules ofdifferent lengths. There may be agents for polymerization, however,which initiate synthesis at the 5′ end and proceed in the otherdirection, using the same process as described above.

[0051] The newly synthesized NMP strand and its complementary nucleicacid strand will form a double-stranded molecule under hybridizingconditions described above and this hybrid is used in subsequent stepsof the process. In the next step, the newly synthesized double-strandedmolecule is subjected to denaturing conditions using any of theprocedures described above to provide single-stranded molecules.

[0052] The above process is repeated on the single-stranded molecules.Additional agent for polymerization, nucleotides, and primers may beadded, if necessary, for the reaction to proceed under the conditionsprescribed above. Again, the synthesis will be initiated at one end ofeach of the oligonucleotide primers and will proceed along the singlestrands of the template to produce additional nucleic acid. After thisstep, half of the extension product will consist of the specific nucleicacid sequence bounded by the two primers.

[0053] The steps of denaturing and extension product synthesis can berepeated as often as needed to amplify the NMP nucleic acid sequence tothe extent necessary for detection. The amount of the specific nucleicacid sequence produced will accumulate in an exponential fashion.

[0054] Sequences amplified by the methods of the invention can befurther evaluated, detected, cloned, sequenced, and the like, either insolution or after binding to a solid support, by any method usuallyapplied to the detection of a specific DNA sequence such as PCR,oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985),allele-specific oligonucleotide (ASO) probe analysis (Conner, et al.,Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligationassays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and thelike. Molecular techniques for DNA analysis have been reviewed(Landegren, et al, Science, 242:229-237, 1988).

[0055] DNA sequences encoding NMP can be expressed in vitro by DNAtransfer into a suitable host cell. “Host cells” are cells in which avector can be propagated and its DNA expressed. The term also includesany progeny of the subject host cell. It is understood that all progenymay not be identical to the parental cell since there may be mutationsthat occur during replication. However, such progeny are included whenthe term “host cell” is used. Methods of stable transfer, in other wordswhen the foreign DNA is continuously maintained in the host, are knownin the art.

[0056] In the present invention, the NMP polynucleotide sequences may beinserted into a recombinant expression vector. The term “recombinantexpression vector” refers to a plasmid, virus or other vehicle known inthe art that has been manipulated by insertion or incorporation of theNMP nucleic acid sequences. Such expression vectors contain a promotersequence which facilitates the efficient transcription of the insertednucleic acid sequence of the host. The expression vector typicallycontains an origin of replication, a promoter, as well as specific geneswhich allow phenotypic selection of the transformed cells. Vectorssuitable for use in the present invention include, but are not limitedto the T7-based expression vector for expression in bacteria (Rosenberget al., Gene 56:125, 1987), the pMSXND expression vector for expressionin mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988) andbaculovirus-derived vectors for expression in insect cells. The DNAsegment can be present in the vector operably linked to regulatoryelements, for example, a promoter (e.g., T7, metallothionein I, orpolyhedrin promoters).

[0057] Polynucleotide sequences encoding NMP can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.Biologically functional viral and plasmid DNA vectors capable ofexpression and replication in a host are known in the art. Such vectorsare used to incorporate DNA sequences of the invention.

[0058] Transformation of a host cell with recombinant DNA may be carriedout by conventional techniques as are well known to those skilled in theart. Where the host is prokaryotic, such as E. coli, competent cellswhich are capable of DNA uptake can be prepared from cells harvestedafter exponential growth phase and subsequently treated by the CaCl₂method by procedures well known in the art. Alternatively, MgCl₂ or RbClcan be used. Transformation can also be performed after forming aprotoplast of the host cell or by electroporation.

[0059] When the host is a eukaryote, such methods of transfection of DNAas calcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with DNA sequences encoding the NMP of the invention, anda second foreign DNA molecule encoding a selectable phenotype, such asthe herpes simplex thymidine kinase gene. Another method is to use aeukaryotic viral vector, such as simian virus 40 (SV40) or bovinepapilloma virus, to transiently infect or transform eukaryotic cells andexpress the protein. (Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982).

[0060] Isolation and purification of microbially expressed protein, orfragments thereof provided by the invention, may be carried out byconventional means including preparative chromatography andimmunological separations involving monoclonal or polyclonal antibodies.Antibodies provided in the present invention are immunoreactive with NMPpolypeptide or fragments thereof.

[0061] The NMP polypeptide of the invention includes fragments andconservative variations of the polypeptides. Minor modifications of theNMP primary amino acid sequence may result in proteins which havesubstantially equivalent activity as compared to the NMP polypeptidedescribed herein. Such modifications may be deliberate, as bysite-directed mutagenesis, or may be spontaneous. All of thepolypeptides produced by these modifications are included herein as longas the biological activity of NMP still exists. Further, deletion of oneor more amino acids can also result in a modification of the structureof the resultant molecule without significantly altering its biologicalactivity. This can lead to the development of a smaller active moleculewhich would have broader utility.

[0062] The term “conservative variation” as used herein denotes thereplacement of an amino acid residue by another, biologically similarresidue. Examples of conservative variations include the substitution ofone hydrophobic residue such as isoleucine, valine, leucine ormethionine for another, or the substitution of one polar residue foranother, such as the substitution of arginine for lysine, glutamic foraspartic acids, or glutamine for asparagine, and the like.

[0063] Peptides of the invention can be synthesized by the well knownsolid phase peptide synthesis methods described Merrifield, J. Am. Chem.Soc., 85:2149, 1962), and Stewart and Young, Solid Phase PeptidesSynthesis, (Freeman, San Francisco, 1969, pp. 27-62), using acopoly(styrene-divinylbenzene) containing 0.1-1.0 mMol amines/g polymer.On completion of chemical synthesis, the peptides can be deprotected andcleaved from the polymer by treatment with liquid HF-10% anisole forabout ¼-1 hours at 0° C. After evaporation of the reagents, the peptidesare extracted from the polymer with 1% acetic acid solution which isthen lyophilized to yield the crude material. This can normally bepurified by such techniques as gel filtration on Sephadex G-15 using 5%acetic acid as a solvent. Lyophilization of appropriate fractions of thecolumn will yield the homogeneous peptide or peptide derivatives, whichcan then be characterized by such standard techniques as amino acidanalysis, thin layer chromatography, high performance liquidchromatography, ultraviolet absorption spectroscopy, molar rotation,solubility, and quantitated by the solid phase Edman degradation.

[0064] The invention includes polyclonal and monoclonal antibodiesimmunoreactive with NMP polypeptide or immunogenic fragments thereof. Ifdesired, polyclonal antibodies can be further purified, for example, bybinding to and elution from a matrix to which NMP polypeptide is bound.Those of skill in the art will know of various other techniques commonin the immunology arts for purification and/or concentration ofpolyclonal antibodies, as well as monoclonal antibodies. Antibody whichconsists essentially of pooled monoclonal antibodies with differentepitopic specificities, as well as distinct monoclonal antibodypreparations are provided. The term antibody or, immunoglobulin as usedin this invention includes intact molecules as well as fragmentsthereof, such as Fab and F(ab′)₂, which are functionally capable ofbinding an epitopic determinant on NMP.

[0065] A preferred method for the identification and isolation ofantibody binding domain which exhibit binding with NMP is thebacteriophage λ vector system. This vector system has been used toexpress a combinatorial library of Fab fragments from the mouse antibodyrepertoire in Escherichia coli (Huse, et al., Science, 246:1275-1281,1989) and from the human antibody repertoire (Mullinax, et al., Proc.Natl. Acad. Sci., 87:8095-8099, 1990). As described therein, receptors(Fab molecules) exhibiting binding for a preselected ligand wereidentified and isolated from these antibody expression libraries. Thismethodology can also be applied to hybridoma cell lines expressingmonoclonal antibodies with binding for a preselected ligand. Hybridomaswhich secrete a desired monoclonal antibody can be produced in variousways using techniques well understood by those having ordinary skill inthe art and will not be repeated here. Details of these techniques aredescribed in such references as Monoclonal Antibodies-Hybridomas: A NewDimension in Biological Analysis, Edited by Roger H. Kennett, et al.,Plenum Press, 1980; and U.S. Pat. No. 4,172,124, incorporated herein byreference.

[0066] As used herein, the term “cell-proliferative disorder” denotesmalignant as well as non-malignant (or benign) disorders. This termfurther encompasses hyperplastic disorders. The cells comprising theseproliferative disorders often appear morphologically and genotypicallyto differ from the surrounding normal tissue. As noted above,cell-proliferative disorders may be associated, for example, withexpression or absence of expression of the NMPs of the invention.Expression of NMP at an inappropriate time during the cell cycle or inan incorrect cell type may result in a cell-proliferative disorder. TheNMP polynucleotide in the form of an antisense polynucleotide is usefulin treating hyperplasia and malignancies of the various organ systems,particularly, for example, those of urogenital origin such as theprostate. In addition, hyperplasia and malignancies of such organs asthe kidney and bladder can be treated using the NMP polynucleotides ofthe invention. Essentially, any disorder which is etiologically linkedto expression of NMP could be considered susceptible to treatment with areagent of the invention which modulates NMP expression. The term“modulate” envisions the suppression of expression of NMP when it isinappropriately expressed or augmentation of NMP expression when it isunder-expressed or when the NMP expressed is a mutant form of thepolypeptide. When a cell-proliferative disorder is associated with NMPexpression, (e.g., BPC-1, 2, 3 and PC-1), such suppressive reagents asantisense NMP polynucleotide sequence or NMP binding antibody can beintroduced to a cell. Alternatively, when a cell proliferative disorderis associated with under-expression or expression of a mutant NMPpolypeptide (e.g., NPB 1-7 and NP-1-3), a sense polynucleotide sequence(the DNA coding strand) or NMP polypeptide can be introduced into thecell.

[0067] The invention provides a method for detecting a cell expressingNMP or a cell proliferative disorder associated with NMP in a subjectcomprising contacting a cell component suspected of expressing NMP orhaving a NMP associated disorder, with a reagent which binds to thecomponent. The cell component can be nucleic acid, such as DNA or RNA,or protein. When the component is nucleic acid, the reagent is a nucleicacid probe or PCR primer. When the cell component is protein, thereagent is an antibody probe. The probes are directly or indirectlydetectably labeled, for example, with a radioisotope, a fluorescentcompound, a bioluminescent compound, a chemiluminescent compound, ametal chelator, or an enzyme. Those of ordinary skill in the art willknow of other suitable labels for binding to the probe or will be ableto ascertain such, using routine experimentation.

[0068] For purposes of the invention, an antibody or nucleic acid probespecific for NMP may be used to detect the presence of NMP polypeptide(using antibody) or polynucleotide (using nucleic acid probe) inbiological fluids or tissues. Oligonucleotide primers based on anycoding sequence region in the NMP sequence are useful for amplifyingDNA, for example by PCR. Any specimen containing a detectable amount ofantigen can be used. A preferred sample of this invention, especiallyfor detecting prostate cancer, is tissue of urogenital origin,specifically tissue of the prostate. Alternatively, biological fluidswhich may contain cells indicative of an NMP-associatedcell-proliferative disorder, such as ejaculate or urine, may be used.Preferably the subject is human.

[0069] Another technique which may also result in greater sensitivityconsists of coupling the probe to low molecular weight haptens. Thesehaptens can then be specifically detected by means of a second reaction.For example, it is common to use such haptens as biotin, which reactswith avidin, or dinitrophenol, pyridoxal, and fluorescein, which canreact with specific antihapten antibodies.

[0070] The method for detecting a cell expressing a particular NMP ofthe invention or a cell-proliferative disorder associated with an NMP,described above, can be utilized for detection of residual prostatecancer or other malignancies or benign hyperplasia conditions in asubject in a state of clinical remission. Additionally, the method fordetecting NMP polypeptide in cells is useful for detecting acell-proliferative disorder by identifying cells expressing specificNMPs in comparison with NMPs expressed in normal cells. Using the methodof the invention, NMP expression can be identified in a cell and theappropriate course of treatment can be employed (e.g., sense orantisense gene therapy, as well as conventional chemotherapy). Since theexpression pattern of the NMPs of the invention vary with the stage ofmalignancy of a cell, a sample such as prostate tissue can be screenedwith a panel of NMP-specific reagents (e.g., nucleic acid probes orantibodies to NMPs) to detect NMP expression and diagnose the stage ofmalignancy of the cell.

[0071] The monoclonal antibodies of the invention are suited for use,for example, in immunoassays in which they can be utilized in liquidphase or bound to a solid phase carrier. In addition, the monoclonalantibodies in these immunoassays can be detectably labeled in variousways. Examples of types of immunoassays which can utilize monoclonalantibodies of the invention are competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antigens using the monoclonalantibodies of the invention can be done utilizing immunoassays which arerun in either the forward, reverse, or simultaneous modes, includingimmunohistochemical assays on physiological samples. Alternatively,antibody of the invention can be used to detect NMPs present inelectrophoretically dispersed gel protocols such as Western blots and2-dimensional gels. Those of skill in the art will know, or can readilydiscern, other immunoassay formats without undue experimentation.

[0072] The monoclonal antibodies of the invention can be bound to manydifferent carriers and used to detect the presence of NMP. Examples ofwell-known carriers include glass, polystyrene, polypropylene,polyethylene, dextran, nylon, amylases, natural and modified celluloses,polyacrylamides, agaroses and magnetite. The nature of the carrier canbe either soluble or insoluble for purposes of the invention. Thoseskilled in the art will know of other suitable carriers for bindingmonoclonal antibodies, or will be able to ascertain such using routineexperimentation.

[0073] In performing the assays it may be desirable to include certain“blockers” in the incubation medium (usually added with the labeledsoluble antibody). The “blockers” are added to assure that non-specificproteins, proteases, or anti-heterophilic immunoglobulins to anti-NMPimmunoglobulins present in the experimental sample do not cross-link ordestroy the antibodies on the solid phase support, or the radiolabeledindicator antibody, to yield false positive or false negative results.The selection of “blockers” therefore may add substantially to thespecificity of the assays described in the present invention.

[0074] It has been found that a number of nonrelevant (i.e.,nonspecific) antibodies of the same class or subclass (isotype) as thoseused in the assays (e.g., IgG1, IgG2a, IgM, etc.) can be used as“blockers”. The concentration of the “blockers” (normally 1-100 μg/μl)is important, in order to maintain the proper sensitivity yet inhibitany unwanted interference by mutually occurring cross reactive proteinsin the specimen.

[0075] As used in this invention, the term “epitope” includes anydeterminant capable of specific interaction with the monoclonalantibodies of the invention. Epitopic determinants usually consist ofchemically active surface groupings of molecules such as amino acids orsugar side chains and usually have specific three dimensional structuralcharacteristics, as well as specific charge characteristics.

[0076] In using the monoclonal antibodies of the invention for the invivo detection of antigen, the detectably labeled monoclonal antibody isgiven in a dose which is diagnostically effective. The term“diagnostically effective” means that the amount of detectably labeledmonoclonal antibody is administered in sufficient quantity to enabledetection of the site having the NMP antigen for which the monoclonalantibody is specific.

[0077] The concentration of detectably labeled monoclonal antibody whichis administered should be sufficient such that the binding to thosecells having NMP is detectable compared to the background. Further, itis desirable that the detectably labeled monoclonal antibody be rapidlycleared from the circulatory system in order to give the besttarget-to-background signal ratio.

[0078] As a rule, the dosage of detectably labeled monoclonal antibodyfor in vivo diagnosis will vary depending on such factors as age, sex,and extent of disease of the individual. The dosage of monoclonalantibody can vary from about 0.001 mg/m² to about 500 mg/m², preferably0.1 mg/m² to about 200 mg/m², most preferably about 0.1 mg/m² to about10 mg/m². Such dosages may vary, for example, depending on whethermultiple injections are given, tumor burden, and other factors known tothose of skill in the art.

[0079] For in vivo diagnostic imaging, the type of detection instrumentavailable is a major factor in selecting a given radioisotope. Theradioisotope chosen must have a type of decay which is detectable for agiven type of instrument. Still another important factor in selecting aradioisotope for in vivo diagnosis is that the half-life of theradioisotope be long enough so that it is still detectable at the timeof maximum uptake by the target, but short enough so that deleteriousradiation with respect to the host is minimized. Ideally, a radioisotopeused for in vivo imaging will lack a particle emission, but produce alarge number of photons in the 140-250 keV range, which may be readilydetected by conventional gamma cameras.

[0080] For in vivo diagnosis, radioisotopes may be bound toimmunoglobulin either directly or indirectly. by using an intermediatefunctional group. Intermediate functional groups which often are used tobind radioisotopes which exist as metallic ions to immunoglobulins arethe bifunctional chelating agents such as diethylenetriaminepentaceticacid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similarmolecules. Typical examples of metallic ions which can be bound to themonoclonal antibodies of the invention are ¹¹¹In, ⁹⁷Ru, ⁶⁷Ga, ⁶⁸Ga,⁷²As, ⁸⁹Zr, and ²⁰¹Tl.

[0081] The monoclonal antibodies of the invention can also be labeledwith a paramagnetic isotope for purposes of in vivo diagnosis, as inmagnetic resonance imaging (MRI) or electron spin resonance (ESR). Ingeneral, any conventional method for visualizing diagnostic imaging canbe utilized. Usually gamma and positron emitting radioisotopes are usedfor camera imaging and paramagnetic isotopes for MRI. Elements which areparticularly useful in such techniques include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Cr,and ⁵⁶Fe.

[0082] The monoclonal antibodies of the invention can be used to monitorthe course of amelioration of NMP associated cell-proliferativedisorder. Thus, by measuring the increase or decrease in the number ofcells expressing a NMP or changes in NMP present in various body fluids,such as ejaculate or urine, it would be possible to determine whether aparticular therapeutic regiment aimed at ameliorating the disorder iseffective.

[0083] The monoclonal antibodies of the invention can also be used,alone or in combination with effector cells (Douillard, et al.Hybridoma, 5 Supp. 1:S139, 1986), for immunotherapy in an animal havinga cell proliferative disorder which expresses NMP polypeptide withepitopes reactive with the monoclonal antibodies of the invention.

[0084] When used for immunotherapy, the monoclonal antibodies of theinvention may be unlabeled or labeled with a therapeutic agent. Theseagents can be coupled either directly or indirectly to the monoclonalantibodies of the invention. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble (Diener, et al., Science, 231:148, 1986) and can beselected to enable drug release from the monoclonal antibody molecule atthe target site. Examples of therapeutic agents which can be coupled tothe monoclonal antibodies of the invention for immunotherapy are drugs,radioisotopes, lectins, and toxins.

[0085] The drugs which can be conjugated to the monoclonal antibodies ofthe invention include non-proteinaceous as well as proteinaceous drugs.The terms “non-proteinaceous drugs” encompasses compounds which areclassically referred to as drugs, for example, mitomycin C,daunorubicin, and vinblastine.

[0086] The proteinaceous drugs with which the monoclonal antibodies ofthe invention can be labeled include immunomodulators and otherbiological response modifiers. The term “biological response modifiers”encompasses substances which are involved in modifying the immuneresponse in such manner as to enhance the destruction of anNMP-associated tumor for which the monoclonal antibodies of theinvention are specific. Examples of immune response modifiers includesuch compounds as lymphokines. Lymphokines include tumor necrosisfactor, the interleukins, lymphotoxin, macrophage activating factor,migration inhibition factor, colony stimulating factor, and interferon.Interferons with which the monoclonal antibodies of the invention can belabeled include alpha-interferon, beta-interferon and gamma-interferonand their subtypes.

[0087] In using radioisotopically conjugated monoclonal antibodies ofthe invention for immunotherapy certain isotypes may be more preferablethan others depending on such factors as tumor cell distribution as wellas isotope stability and emission. If desired, the tumor celldistribution can be evaluated by the in vivo diagnostic techniquesdescribed above. Depending on the cell proliferative disease someemitters may be preferable to others. In general, alpha and betaparticle-emitting radioisotopes are preferred in immunotherapy. Forexample, if an animal has solid tumor foci a high energy beta emittercapable of penetrating several millimeters of tissue, such as ⁹⁰ Y, maybe preferable. On the other hand, if the cell proliferative disorderconsists of simple target cells, as in the case of leukemia, a shortrange, high energy alpha emitter, such as ²¹²Bi, may be preferable.Examples of radioisotopes which can be bound to the monoclonalantibodies of the invention for therapeutic purposes are ¹²⁵I, ¹³¹I,⁹⁰Y, ⁶⁷Cu, ²¹²Bi, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, ⁶⁵Zn, and ¹⁸⁸Re.

[0088] Lectins are proteins, usually isolated from plant material, whichbind to specific sugar moieties. Many lectins are also able toagglutinate cells and stimulate lymphocytes. However, ricin is a toxiclectin which has been used immunotherapeutically. This is preferablyaccomplished by binding the alpha-peptide chain of ricin, which isresponsible for toxicity, to the antibody molecule to enable sitespecific delivery of the toxic effect.

[0089] Toxins are poisonous substances produced by plants, animals, ormicroorganisms, that, in sufficient dose, are often lethal. Diphtheriatoxin is a substance produced by Corynebacterium diphtheria which can beused therapeutically. This toxin consists of an alpha and beta subunitwhich under proper conditions can be separated. The toxic A componentcan be bound to an antibody and used for site specific delivery to a NMPbearing cell. Other therapeutic agents which can be coupled to themonoclonal antibodies of the invention are known, or can be easilyascertained, by those of ordinary skill in the art.

[0090] The labeled or unlabeled monoclonal antibodies of the inventioncan also be used in combination with therapeutic agents such as thosedescribed above. Especially preferred are therapeutic combinationscomprising the monoclonal antibody of the invention and immunomodulatorsand other biological response modifiers.

[0091] Thus, for example, the monoclonal antibodies of the invention canbe used in combination with alpha-interferon. This treatment modalityenhances monoclonal antibody targeting of carcinomas by increasing theexpression of monoclonal antibody reactive antigen by the carcinomacells (Greiner, et al., Science, 235:895, 1987). Alternatively, themonoclonal antibody of the invention could be used, for example, incombination with gamma-interferon to thereby activate and increase theexpression of Fc receptors by effector cells which, in turn, results inan enhanced binding of the monoclonal antibody to the effector cell andkilling of target tumor cells. Those of skill in the art will be able toselect from the various biological response modifiers to create adesired effector function which enhances the efficacy of the monoclonalantibody of the invention.

[0092] When the monoclonal antibody of the invention is used incombination with various therapeutic agents, such as those describedherein, the administration of the monoclonal antibody and thetherapeutic agent usually occurs substantially contemporaneously. Theterm “substantially contemporaneously” means that the monoclonalantibody and the therapeutic agent are administered reasonably closetogether with respect to time. Usually, it is preferred to administerthe therapeutic agent before the monoclonal antibody. For example, thetherapeutic agent can be administered 1 to 6 days before the monoclonalantibody. The administration of the therapeutic agent can be daily, orat any other interval, depending upon such factors, for example, as thenature of the tumor, the condition of the patient and half-life of theagent.

[0093] Using monoclonal antibodies of the invention, it is possible todesign therapies combining all of the characteristics described herein.For example, in a given situation it may be desirable to administer atherapeutic agent, or agents, prior to the administration of themonoclonal antibodies of the invention in combination with effectorcells and the same, or different, therapeutic agent or agents. Forexample, it may be desirable to treat patients with prostate, kidney orbladder carcinoma by first administering gamma-interferon andinterleukin-2 daily for 3 to 5 days, and on day 5 administer themonoclonal antibody of the invention in combination with effector cellsas well as gamma-interferon, and interleukin-2.

[0094] It is also possible to utilize liposomes with the monoclonalantibodies of the invention in their membrane to specifically deliverthe liposome to the tumor expressing NMP. These liposomes can beproduced such that they contain, in addition to the monoclonal antibody,such immunotherapeutic agents as those described above which would thenbe released at the tumor site (Wolff, et al., Biochemical et BiophysicalActa, 802:259, 1984).

[0095] The dosage ranges for the administration of monoclonal antibodiesof the invention are those large enough to produce the desired effect inwhich the symptoms of the malignant disease are ameliorated. The dosageshould not be so large as to cause adverse side effects, such asunwanted cross-reactions, anaphylactic reactions, and the like.Generally, the dosage will vary with the age, condition, sex and extentof the disease in the patient and can be determined by one of skill inthe art. The dosage can be adjusted by the individual physician in theevent of any complication. Dosage can vary from about 0.1 mg/kg to about2000 mg/kg, preferably about 0.1 mg/kg to about 500 mg/kg, in one ormore dose administrations daily, for one or several days. Generally,when the monoclonal antibodies of the invention are administeredconjugated with therapeutic agents, lower dosages, comparable to thoseused for in vivo diagnostic imaging, can be used.

[0096] The monoclonal antibodies of the invention can be administeredparenterally by injection or by gradual perfusion over time. Themonoclonal antibodies of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, or transdermally, alone or in combination with effectorcells.

[0097] The present invention also provides a method for treating asubject with an NMP-associated cell-proliferative disorder using an NMPnucleotide sequence. An NMP nucleotide sequence which may encode asuppressor polypeptide may be under-expressed as compared to expressionin a normal cell, therefore it is possible to design appropriatetherapeutic or diagnostic techniques directed to this sequence. Thus,where a cell-proliferative disorder is associated with the expression ofan NMP associated with malignancy, nucleic acid sequences that interferewith NMP expression at the translational level can be used. Thisapproach utilizes, for example, antisense nucleic acid and ribozymes toblock translation of a specific NMP mRNA, either by masking that mRNAwith an antisense nucleic acid or by cleaving it with a ribozyme. Incases when a cell proliferative disorder or abnormal cell phenotype isassociated with the under expression of NMP suppressor for example,nucleic acid sequences encoding NMP (sense) could be administered to thesubject with the disorder.

[0098] Antisense nucleic acids are DNA or RNA molecules that arecomplementary to at least a portion of a specific mRNA molecule(Weintraub, Scientific American, 262:40, 1990). In the cell, theantisense nucleic acids hybridize to the corresponding mRNA, forming adouble-stranded molecule. The antisense nucleic acids interfere with thetranslation of the mRNA since the cell will not translate a mRNA that isdouble-stranded. Antisense oligomers of about 15 nucleotides arepreferred, since they are easily synthesized and are less likely tocause problems than larger molecules when introduced into the targetNMP-producing cell. The use of antisense methods to inhibit the in vitrotranslation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).

[0099] Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences which encode these RNAs, it is possible to engineer moleculesthat recognize specific nucleotide sequences in an RNA molecule andcleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantageof this approach is that, because they are sequence-specific, only mRNAswith particular sequences are inactivated.

[0100] There are two basic types of ribozymes namely, tetrahymena-type(Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type.Tetrahymena-type ribozymes recognize sequences which are four bases inlength, while “hammerhead”-type ribozymes recognize base sequences 11-18bases in length. The longer the recognition sequence, the greater thelikelihood that that sequence will occur exclusively in the target mRNAspecies. Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating a specific mRNA species and18-based recognition sequences are preferable to shorter recognitionsequences.

[0101] The present invention also provides gene therapy for thetreatment of cell proliferative disorders which are mediated by NMP.Such therapy would achieve its therapeutic effect by introduction of theappropriate NMP polynucleotide (antisense or sense), into cells ofsubjects having the proliferative disorder. Delivery of antisense NMPpolynucleotide can be achieved using a recombinant expression vectorsuch as a chimeric virus or a colloidal dispersion system. Disordersassociated with under-expression of an NMP or expression of acancer-associated NMP, could be treated using gene therapy with sense orantisense nucleotide sequences, respectively.

[0102] Various viral vectors which can be utilized for gene therapy astaught herein include adenovirus, herpes virus, vaccinia, or,preferably, an RNA virus such as a retrovirus. Preferably, theretroviral vector is a derivative of a murine or avian retrovirus.Examples of retroviral vectors in which a single foreign gene can beinserted include, but are not limited to: Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additionalretroviral vectors can incorporate multiple genes. All of these vectorscan transfer or incorporate a gene for a selectable marker so thattransduced cells can be identified and generated. By inserting a NMPsequence of interest into the viral vector, along with another genewhich encodes the ligand for a receptor on a specific target cell, forexample, the vector is now target specific. Retroviral vectors can bemade target specific by inserting, for example, a polynucleotideencoding a sugar, a glycolipid, or a protein. Preferred targeting isaccomplished by using an antibody to target the retroviral vector. Thoseof skill in the art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the NMP sense or antisense polynucleotide.

[0103] Since recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided, for example, by using helper cell lines thatcontain plasmids encoding all of the structural genes of the retrovirusunder the control of regulatory sequences within the LTR. These plasmidsare missing a nucleotide sequence which enables the packaging mechanismto recognize an RNA transcript for encapsidation. Helper cell lineswhich have deletions of the packaging signal include but are not limitedto ψ2, PA317 and PA 12, for example. These cell lines produce emptyvirions, since no genome is packaged. If a retroviral vector isintroduced into such cells in which the packaging signal is intact, butthe structural genes are replaced by other genes of interest, the vectorcan be packaged and vector virion produced.

[0104] Alternatively, NIH 3T3 or other tissue culture cells can bedirectly transfected with plasmids encoding the retroviral structuralgenes gag, pol and env, by conventional calcium phosphate transfection.These cells are then transfected with the vector plasmid containing thegenes of interest. The resulting cells release the retroviral vectorinto the culture medium.

[0105] Another targeted delivery system for NMP antisensepolynucleotides a colloidal dispersion system. Colloidal dispersionsystems include macromolecule complexes, nanocapsules, microspheres,beads, and lipid-based systems including oil-in-water emulsions,micelles, mixed micelles, and liposomes. The preferred colloidal systemof this invention is a liposome. Liposomes are artificial membranevesicles which are useful as delivery vehicles in vitro and in vivo. Ithas been shown that large unilamellar vesicles (LUV), which range insize from 0.2-4.0 um can encapsulate a substantial percentage of anaqueous buffer containing large macromolecules. RNA, DNA and intactvirions can be encapsulated within the aqueous interior and be deliveredto cells in a biologically active form (Fraley, et al., Trends Biochem.Sci., 6:77, 1981). In addition to mammalian cells, liposomes have beenused for delivery of polynucleotides in plant, yeast and bacterialcells. In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino, et al., Biotechniques, 6:682, 1988).

[0106] The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, especiallycholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations.

[0107] Examples of lipids useful in liposome production includephosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phos-phatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipaimitoylphosphatidylcholine and distearoylphosphatidylcholine.

[0108] The targeting of liposomes has been classified based onanatomical and mechanistic factors. Anatomical classification is basedon the level of selectivity, for example, organ-specific, cell-specific,and organelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

[0109] The surface of the targeted delivery system may be modified in avariety of ways. In the case of a liposomal targeted delivery system,lipid groups can be incorporated into the lipid bilayer of the liposomein order to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

[0110] In general, the compounds bound to the surface of the targeteddelivery system will be ligands and receptors which will allow thetargeted delivery system to find and “home in” on the desired cells. Aligand may be any compound of interest which will bind to anothercompound, such as a receptor.

[0111] In general, surface membrane proteins which bind to specificeffector molecules are referred to as receptors. In the presentinvention, antibodies of the invention are preferred receptors.Antibodies can be used to target liposomes to specific cell-surfaceligands, in this case the NMPs of choice. Preferably, the target tissueis urogenital and specifically is prostate tissue. Kidney and bladdertissue may also be utilized. A number of procedures can be used tocovalently attach either polyclonal or monoclonal antibodies to aliposome bilayer. Antibody-targeted liposomes can include monoclonal orpolyclonal antibodies or fragments thereof such as Fab, or F(ab′)₂, aslong as they bind efficiently to an the antigenic epitope on the targetcells.

[0112] Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's intravenousvehicles include fluid and nutrient replenishers, electrolytereplenishers (such as those based on Ringer's dextrose), and the like.Preservatives and other additives may also be present such as, forexample, antimicrobials, anti-oxidants, chelating agents and inert gasesand the like.

[0113] The invention also relates to a method for preparing a medicamentor pharmaceutical composition comprising the polynucleotides or themonoclonal antibodies of the invention, the medicament being used fortherapy of NMP associated cell proliferative disorders.

[0114] The NMPs of the invention are useful as a screening tool forcompositions which affect an NMP of a cell. Thus, in another embodiment,the invention provides a method for identifying a composition whichaffects an NMP comprising incubating the components, which include thecomposition to be tested and the cell (or cell suspension), underconditions sufficient to allow the components to interact, thensubsequently measuring the effect of the composition on the NMP. Theobserved effect on the NMP may be either inhibitory or stimulatory.

[0115] For example, in a malignant cell of the prostate, compositionswhich are inhibitory to BPC-1, BPC-2, BPC-3, or PC-1 expression can beidentified by measuring the level of the NMP in the cell or cell extractbefore and after treatment with the composition. Alternatively, thelevels of NP-1, NP-2, or NP-3 can be monitored to identify compositionswhich stimulate expression of these NMPs, found in normal cells.

[0116] The material for use in the assay of the invention are ideallysuited for the preparation of a kit. Such a kit may comprise a carriermeans being compartmentalized to receive in close confinement one ormore container means such as vials, tubes, and the like, each of thecontainer means comprising one of the separate elements to be used inthe method.

[0117] For example, one of the container means may comprise a probewhich is or can be detectably labelled. Such probe may be an antibody ornucleotide specific for a target protein or a target nucleic acid,respectively, wherein the target is indicative, or correlates with, thepresence of an NMP of the invention. Where the kit utilizes nucleic acidhybridization to detect the target nucleic acid, the kit may also havecontainers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradionuclide label.

[0118] The following Examples are intended to illustrate, but not tolimit the invention. While such Examples are typical of those that mightbe used, other procedures known to those skilled in the art mayalternatively be utilized.

EXAMPLE 1 IDENTIFICATION AND PURIFICATION OF NUCLEAR MATRIX PROTEINS

[0119] Patients.

[0120] Fresh prostatic tissue was studied from 21 men undergoing radicalretropubic prostatectomy for clinically localized (Stage B, T2) prostatecancer (N-19) (Gleason grade 5-9) or open prostatectomy for benignprostatic hyperplasia (BPH, N=2).

[0121] Tissue Preparation.

[0122] Fresh tissue was obtained within 15 minutes of surgical removal.Approximately one gram of gross tumor was taken from a palpable tumornodule from 14 specimens. One gram of normal prostate tissue wasobtained from the prostatic lobe contralateral to the tumor nodule in 13specimens. One gram of BPH tissue was obtained from the periurethralregion of the contralateral lobe in 12 specimens and 25-30 grams fromeach of the 2 open prostatectomy specimens. All tissues removed werehistologically confirmed with hematoxylin and eosin sections on both theproximal and distal ends of the section.

[0123] Purification of Nuclear Matrix Proteins.

[0124] Nuclear matrix proteins were isolated according to themethodology of Fey and Penman (Proc. Natl. Acad. Sci., USA, 85:121-125,1988). Briefly, fresh human prostate tissue was minced into small (1mm³) pieces and homogenized with a Teflon pestle on ice with 0.5% TritonX-100 in a solution containing 2 mM vanadyl ribonucleoside (RNAaseinhibitor) containing 1 mM phenylmethylsulfonyl fluoride (serineprotease inhibitor) to release the lipids and soluble proteins. Extractswere then filtered through a 350 micron nylon mesh and extracted with0.25 M ammonium sulfate to release the soluble cytoskeletal elements.DNase treatment at 25° C. was used to remove the soluble chromatin. Theremaining fraction contained intermediate filaments and nuclear matrixproteins. This fraction was then disassembled with 8 M urea, and theinsoluble components, which consist principally of carbohydrates andextracellular matrix components, were pelleted. The urea was dialyzedout and the intermediate filaments allowed to reassemble and wereremoved by centrifugation. The nuclear matrix proteins were then ethanolprecipitated. Protein concentrations were determined with the Comassie®Plus protein assay reagent kit (Pierce, Rockford, Ill.) with bovineserum albumin as a standard. For preparation for gel electrophoresis,the nuclear matrix proteins were redissolved in a sample bufferconsisting of 9 M urea, 65 mM 3-[(3-cholamidopropyl)dimethylamino)-1-propanosulfonate, 2.2% ampholytes and 140 mMdithiothreitol.

[0125] Two-Dimensional Electrophoresis.

[0126] High resolution two-dimensional gel electrophoresis was carriedout utilizing the Investigator 2-D gel system (Milligan/Biosearch,Bedford, Mass.) (Patton, W. F., et al., BioTechniques, 8:518-527, 1990.One-dimensional isoelectric focusing was carried out for 18,000 V-husing 1 nm×18 on tube gels after 1.5 hours of prefocusing. The tube gelswere extruded and placed on top of 1 -mm pre-cast 10% Tris-acetatesodium dodecyl sulfate Duracryl™ (Millipore, Colo., Bedford, Mass.) hightensile strength (HTS) polyacrylamide electrophoresis slab gels and thegels were electrophoresed with 12° C. constant temperature regulationfor approximately 5 hours. Gels were fixed with 50% methanol and 10%acetic acid. After thorough rinsing and rehydration, gels were treatedwith 5% glutaraldehyde and 5 mM dithiothreitol after buffering with 50Mm phosphate (pH 7.2). Gels were stained with silver stain using themethodology of Wray (Wray, W. et al., Anal. Biochem, 118:197-203, 1981)(Accurate Chemical Co., Inc., Westbury, N.Y.). Fifty micrograms ofnuclear matrix protein were loaded for each gel. Protein molecularweight standards were determined with the GELCODE (Dacheng, H., et al.,J. Cell Biol., 110:569-580, 1990), protein molecular weight market kit(MW 12,400-97,400), (Pierce, Rockford, Ill.). Isoelectric points weredetermined using carbamylated creatine kinase standards (pH 7.0-4.950,(BDH Limited, England)). Only protein spots clearly and reproduciblyobserved or absent in all samples from the various tissues wereconsidered when determining variations in nuclear matrix proteinsbetween tissues.

[0127] There was marked similarity seen in the nuclear matrix proteinpatterns between patients with approximately 120 of 150 proteins spotsconsistently seen from patient to patient. Fourteen nuclear matrixproteins were identified that were consistently present or absent whencomparing normal prostate BPH and prostate cancer. A protein (PC-1),with a molecular weight of 56 Kd and pl=6.58, represented a nuclearmatrix protein that appeared in all (14/14) human prostate cancerspecimens studied but was not detected in any normal prostate (0/13) orBPH tissue (0/14).

[0128]FIG. 1 shows nuclear matrix protein composition of normal humanprostate (A), benign prostatic hyperplasia (B) and prostate cancer (C).Four proteins known to be present in most nuclear matrix preparations,lamin A, lamin B, lamin C and actin were identified based uponpreviously reported molecular weights and isoelectric points (Fey, E.G., et al., Proc. Natl. Acad. Sci. USA, 85:121-125, 1988) and labeled onFIG. 1A as LA, LB, LC and A respectively.

[0129]FIG. 1 demonstrates the typical high resolution two-dimensionalgel electrophoresis patterns for nuclear matrix proteins isolated fromnormal human prostate (FIG. 1A), human BPH (FIG. 1B) and human prostatecancer (FIG. 1C). Gel spots differing between normal prostate, BPH andprostate cancer (in all specimens examined) have been marked with arrowsand identified with labels corresponding to those in Table 1. Table 1demonstrates the molecular weight and isoelectric points of the 14different protein spots found to be consistently present or absent whencomparing nuclear matrix proteins from normal prostate, BPH and prostatecancer tissue for this group of 21 patients.

[0130]FIG. 2 summarizes the location of the protein spots that differedbetween the various tissues and shows specific nuclear matrix proteinsin BPH and prostate cancer. Schematic of major tissue specific nuclearmatrix proteins of normal prostate, BPH and prostate cancer.Abbreviations: kD—molecular weight in thousands, SDS-PAGE—sodium dodecylsulfate-polyacrylamide gel electrophoresis and pl—isoelectric point andBPH—benign prostatic hyperplasia.

[0131] No NMPs were detected which were present only in BPH and wereabsent in normal prostate and prostate cancer. Likewise, no NMP's werepresent both in normal prostate and prostate cancer, but were absent inBPH. PC-1 (molecular weight 56 Kd and isoelectric point 6.58) representsan NMP seen only in human prostate cancer tissue and was consistentlyabsent in all normal prostate and BPH samples. In additional testing,PC-1 was found in kidney and bladder cancer specimens, but was notdetected in normal kidney or bladder tissue.

[0132] The absence of NPB-1-7 and NP-1-3 in malignant cells suggeststhat genes encoding these NMPs may function as cancer suppressor genes.This is especially true with respect to NP-1-3 which were also absent inbenign hyperplastic tissue. TABLE 1 NUCLEAR MATRIX PROTEINS FROM FRESHNORMAL PROSTATE, BPH AND PROSTATE CANCER TISSUE Normal BPH CancerProtein M_(r) pl (N = 13) (N = 14) (N = 14) NPB-1 17,000 6.91 + + −NPB-2 17,000 8.30 + + − NPB-3 12,000 8.40 + + − NPB-4 12,000 6.91 + + −NPB-5 43,000 6.27 + + − NPB-6 43,000 6.22 + + − NPB-7 43,000 6.14 + + −NP-1 12,000 7.50 + − − NP-2 11,500 7.62 + − − NP-3 11,000 8.30 + − −BPC-1 42,500 5.80 − + + BPC-2 42,000 5.73 − + + BPC-3 41,000 5.64 − + +PC-1 56,000 6.58 − − +

[0133] The designation of each protein corresponds to the identifiedproteins in FIG. 1.

EXAMPLE 2 MODELS FOR PROGRESSION FROM NORMAL PROSTATE CELLS TO PROSTATICCANCER

[0134] Although the precise molecular and/or environmental eventsnecessary for the development of prostatic disease are largely unknown,it has been well established that the development of prostate cancer isa multistep process (Carter, H. B., et al., J. Urol., 143:742-746,1990). Epidemiologic studies based on the original work of Ashley(Ashley, D. F. B., J. Path. Bact, 90:217-225, 1965) and Armitage andDoll (Armitage P. and Doll, R., Brit. J. Cancer, 8:1-15, 1954) using agespecific incidence rates for prostate cancer and BPH in the U.S.demonstrate that development of BPH is most likely a two-step processwhile the development of clinically evident prostate cancer most likelyinvolves a multi-step (greater than 2 event) process (Carter, H. B., etal., J. Urol., 143:742-746, 1990).

[0135] Two different models can be postulated for the progression of anormal prostatic epithelial cell to either BPH or prostate cancer. FIG.3 shows two models of multistep progression from normal prostate(Normal) to benign prostatic hyperplasia (BPH) or to prostate cancer(Cancer). Model I predicts that similar events occur in both pathways.Model II predicts different events occurring when progressing fromnormal to BPH as when progressing to cancer. The first model (Model I)predicts that the early events for progression from either normal to BPHor normal to prostate cancer are similar (events A-B in Model I). Thesecond model (Model II) predicts that progression for BPH and cancerwould undergo different events (events A-B versus events E-H). Using thepresence or absence of nuclear matrix proteins as a phenotypic marker totest these models, it would be predicted that Model I would be satisfiedif a specific group of protein spots were either absent or present inboth BPH and prostate cancer (NP 1-3, BPC 1-3) and additional proteinspots were present or absent in only prostate cancer (NPB 1-7 or PC-1).Thus all of the differences observed in the nuclear matrix proteinssatisfied Model I. In order to satisfy Model II, a protein(s) need bepresent or absent in BPH only and this was not observed in any samples.Thus, these data support Model I in which similar phenotypic expressionsare occurring in the nuclear matrix of cells progressing to BPH as thosecells progressing to prostate cancer. As a result, the NMPs of theinvention can be used to monitor and detect the stage and progression ofa cell proliferative disorder, such as prostate cancer, from normalcy tobenign disease to malignancy. This information, in turn, can be used toinitiate appropriate therapy.

Example 3 PC-1 Isolation and Microsequencing

[0136] Isolation of “PC-1 Spots” from 2-D Gels:

[0137] LNCap cells were grown in 10-stage factories by a modification ofstandard conditions, and harvested by centrifugation. The nuclear matrixprotein (NMP) fraction was isolated from these cells by the method ofFey and Penman (Proc. Nat'l. Acad. Sci. USA, 85: 121-125, 1988, withmodifications, 200 μg aliquots of the NMP fraction was fractionated bytwo-dimensional (2-D) electrophoresis essentially as described inKeesee, et al (Proc. Nat'l. Acad. Sci. USA, 91:1913-1916, 1994, with 10%Duracryl gels in the second dimension.

[0138] Following electrophoresis, proteins were visualized by the“negative-staining” protocol described in Ferandez-Patron, et al.,BioTechniques 12:564-573, 1992. The spot described as “PC-1” by Partin,et al. (Cancer Res.53:744-746, 1993) was identified on each gel by itsobserved size and isolectric point, and its position within theconstellation of 2-D gel spots routinely observed. The PC-1 spots wereexcised with a polypropylene pipettor tip and destained briefly in 2%citric acid.

[0139] Concentration of PC-1 from Gel Spots:

[0140] The destained gel spots were equilibrated with 100 mM Tris, pH7.4, then with 1x sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) sample buffer, and 20-60 spots were pooledand re-electrophoresed (to further concentrate the protein) on anadditional 10% SDS-PAGE minigel. These gels were electroblotted ontoImmobilon PVDF and stained with Buffalo Black as described in Obar andHolzbaur (Methods in Cell Biol. 37:361-405, 1994). The stained PC-1bands were excised with a razor blade, and stored wet. The quantity oftotal protein in these stained bands was determined by solid phase aminoacid analysis.

[0141] Fragmentation and Sequencing:

[0142] PVDF-immobilized bands comprising four hundred eleven PC-1 gelspots (estimated as ˜56 mol total protein) were pooled and subjected totrypsin digestion and HPLC fragment separation at the MicrochemistryFacility of the Worcester Foundation for Experimental Biology,essentially by the method of Fernandez, et al. (Anal. Biochem.218:112-117, 1994). Twenty four fragments were isolated as reverse phasepeak fractions, and two of these, #9 and #12, were selected formicrosequencing. The resulting amino-terminal amino acid sequences ofthe fragments were: Peak #9: (NH₃)-G R P V G F P M R G R G G F D-(CO₂)(SEQ ID NO:1) Peak #12: (NH₃)-G S Y G D L G G P I I T T Q V T I PK-(CO₂) (SEQ ID NO:2)

[0143] Probing of Sequence Database:

[0144] Each of these sequences was used to probe the current version ofthe non-redundant protein sequence database at the National Center forBiomedical Instrumentation (NCBI). Both peptides matched exactly withthe amino terminal sequences of predicted tryptic peptides from thehuman, rat, and mouse homologs of the same protein, heterogenous nuclearribonucleoprotein K (hnRNP K). As the probabilities of each of these twopeptide sequences matching exactly with any given database entry bychance were calculated to be 4.2×10⁻⁶ and 1.8×10⁻⁷, respectively, it wasestimated that the probability of both sequences matching differentregions of the same database entry by chance as less than 7.6×10−13.Thus we conclude that PC-1 is a product of the same gene as the hnRNP Kpolypeptide, and as the parent LNCap cell line is of human prostateorigin, PC-1 is likely an isoform or a degradation product of humanhnRNP K.

[0145] The foregoing is meant to illustrate, but not to limit, the scopeof the invention. Indeed, those of ordinary skill in the art can readilyenvision and produce further embodiments, based on the teachings herein,without undue experimentation.

1 2 15 amino acids amino acid Not Relevant linear protein 1 Gly Arg ProVal Gly Phe Pro Met Arg Gly Arg Gly Gly Phe Asp 1 5 10 15 19 amino acidsamino acid Not Relevant linear protein 2 Gly Ser Tyr Gly Asp Leu Gly GlyPro Ile Ile Thr Thr Gln Val Thr 1 5 10 15 Ile Pro Lys

1. A method for detecting a cell proliferative disorder in a subjectcomprising contacting a cellular component with a reagent which binds toPC-1 (prostate cancer-1) protein.
 2. The method of claim 1, wherein thecell proliferative disorder is in urogenital tissue.
 3. The method ofclaim 2, wherein the urogenital tissue is the prostate.
 4. The method ofclaim 1, wherein the cellular component is the nucleic acid.
 5. Themethod of claim 4, wherein the nucleic acid is DNA.
 6. The method ofclaim 4, wherein the nucleic acid is RNA.
 7. The method of claim 1,wherein the reagent is a probe.
 8. The method of claim 7, wherein thereagent is nucleic acid.
 9. The method of claim 7, wherein the probe isdetectably labeled.
 10. The method of claim 9, wherein the label isselected from the group consisting of a radioisotope, a bioluminescentcompound, a chemiluminescent compound, a fluorescent compound, a metalchelate, or an enzyme.